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Therapeutic applications of laminin and laminin-derived protein fragments

a technology of laminin and laminin-derived protein, which is applied in the field of discovery, identification and use of laminin, laminin-derived protein fragments, and laminin-derived polypeptides, can solve the problems of neuronal cell death, toxicity and neuronal cell death, and the fact that this potential fibrillar protein remains soluble in circulating biological fluids

Inactive Publication Date: 2008-01-01
UNIV OF WASHINGTON
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about the discovery and use of laminin and its fragments as therapeutic agents and diagnostic tools for Alzheimer's disease and other amyloid diseases. Laminin is a component of the basement membrane that has been found to interact with beta-amyloid protein, which is associated with Alzheimer's disease and other amyloid diseases. The invention provides new methods for treating and diagnosing these diseases using laminin and its fragments. The invention also identifies a specific region within laminin that interacts with beta-amyloid protein, which can be used for diagnosis and treatment. The invention also describes the presence of laminin fragments in human biological fluids, such as serum and cerebrospinal fluid, which can be used for diagnosis and therapeutic applications. Overall, the invention provides new tools for preventing and treating amyloid diseases and advancing the diagnosis of these diseases.

Problems solved by technology

Fibrillar Aβ amyloid deposition in Alzheimer's disease is believed to be detrimental to the patient and eventually leads to toxicity and neuronal cell death, characteristic hallmarks of Alzheimer's disease.
However, it is not known why this potential fibrillar protein remains soluble in circulating biological fluids.

Method used

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  • Therapeutic applications of laminin and laminin-derived protein fragments
  • Therapeutic applications of laminin and laminin-derived protein fragments
  • Therapeutic applications of laminin and laminin-derived protein fragments

Examples

Experimental program
Comparison scheme
Effect test

example 1

Binding of Laminin to the Beta-Amyloid Protein (Aβ) of Alzheimer's Disease

[0073]2 μg of Aβ (1-40)(Bachem Inc., Torrance, Calif. USA; Lot #WM365) in 40 μl of Tris-buffered saline (TBS)(pH 7.0) was allowed to bind overnight at 4° C. to microtiter wells (Nunc plates, Maxisorb). The next day all of the microtiter wells were blocked by incubating with 300 μl of Tris-buffered saline containing 100 mM Tris-HCl, 50 MM NaCl, 0.05% Tween-20, and 3 mM NaN3 (pH 7.4)(TTBS) plus 2% bovine serum albumin (BSA). Various dilutions (ie. 1:10, 1:30, 1:90, 1:270, 1:810, 1:2430 and 1:7290) of Engelbreth-Holm-Swarm (EHS) mouse tumor laminin (1 mg / ml)(Sigma Chemical Co., St. Louis, Mo., USA) in 250 μl of TBS (pH 7.4) were placed in wells (in triplicate) either containing substrate bound Aβ (1-40) or blank, and allowed to bind overnight at 4° C. overnight. The next day, the wells were rinsed 3 times with TTBS, and then probed for 2 hours with 100 μl of rabbit anti-laminin antibody (Sigma Chemical Company, S...

example 2

Inhibition of Alzheimer's Disease Aβ Fibril Formation by Laminin

[0077]The effects of laminin on Aβ fibrillogenesis was also determined using the previously described method of Thioflavin T fluorometry (Naiki et al, Lab. Invest. 65:104-110, 1991; Levine III, Protein Sci. 2:404-410, 1993; Levine III, Int. J. Exp. Clin. Invest. 2:1-6, 1995; Naiki and Nakakuki, Lab. Invest. 74:374-383, 1996). In this assay, Thioflavin T binds specifically to fibrillar amyloid and this binding produces a fluorescence enhancement at 480 nm that is directly proportional to the amount of amyloid fibrils formed (Naiki et al, Lab. Invest. 65:104-110, 1991; Levine III, Protein Sci. 2:404-410, 1993; Levine III, Int. J. Exp. Clin. Invest. 2:1-6, 1995; Naiki and Nakakuki, Lab. Invest. 74:374-383, 1996). In a first study, the effects of EHS laminin on Aβ (1-40) fibrillogenesis was assessed. For this study, 25 μM of freshly solubilized Aβ (1-40)(Bachem Inc., Torrance, Calif., USA; Lot #WM365) was incubated in micro...

example 3

Laminin Causes Dose-Dependent Dissolution of Pre-Formed Alzheimer's Disease Amyloid Fibrils

[0082]The next study was implemented to determine whether laminin was capable of causing a dose-dependent dissolution of pre-formed Alzheimer's disease Aβ (1-40) amyloid fibrils. This type of activity would be important for any potential anti-Alzheimer's amyloid drug which can be used in patients who already have substantial amyloid deposition in brain. For example, Alzheimer's disease patients in mid-to late stage disease have abundant amyloid deposits in their brains as part of both neuritic plaques and cerebrovascular amyloid deposits. A therapeutic agent capable of causing dissolution of pre-existing amyloid would be advantageous for use in these patients who are at latter stages of the disease process.

[0083]For this study, 1 mg of Aβ (1-40)(Bachem Inc., Torrance, Calif., USA; Lot #WM365) was dissolved in 1.0 ml of double distilled water (1 mg / ml solution) and then incubated at 37° C. for ...

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Abstract

Laminin and specific laminin-derived protein fragments are disclosed as potent inhibitors of Alzheimer's disease type amyloidoses. A specific region is identified within laminin which interacts with the Alzheimer's disease beta-amyloid protein and contributes to the observed inhibitory and therapeutic effects.A prominent ˜130 kilodalton band in laminin was found in human serum and cerebrospinal fluid which primarily interacted with Aβ as determined by ligand blotting methodology. This ˜130 kilodalton laminin fragment is known as the E8 fragment and is also believed to consist of the globular domains of the laminin A chain. The interaction of specific laminin fragments such as the newly discovered ˜130 kDa protein is believed to bind Aβ in biological fluids and keep it in a soluble state.

Description

[0001]This is a continuation of U.S. Application Ser. No. 08 / 947,057 filed Oct. 8, 1997 now abandoned, which claims priority to U.S. Provisional Application No. 60 / 027,981 filed Oct. 8, 1996.TECHNICAL FIELD [0002]The invention relates to the discovery, identification and use of laminin, laminin-derived protein fragments, and laminin-derived polypeptides, as well as related peptides and antibodies, for the therapeutic intervention and diagnosis of Alzheimer's disease and other amyloidoses. In addition, the discovery and identification of an Alzheimer's beta-amyloid protein (Aβ) specific binding region within the globular domain repeats of the laminin A chain, has led to new diagnostic and therapeutic applications for Alzheimer's disease and other amyloidoses which are disclosed.BACKGROUND OF THE INVENTION[0003]Alzheimer's disease is characterized by the accumulation of a 39-43 amino acid peptide termed the beta-amyloid protein or Aβ, in a fibrillar form, existing as extracellular amy...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): G01N33/567G01N33/566A61K38/00C07K1/00C07K14/00C07K17/00C12N5/00C12N5/02C12N5/08C07K14/78
CPCC07K14/78A61K38/00
Inventor CASTILLO, GERARDOSNOW, ALAN D.
Owner UNIV OF WASHINGTON
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